PROJECT SUMMARY Dietary amino acid insufficiency (AAI) can yield a wide variety of outcomes that can be either adverse or beneficial to health. A clearer understanding of the molecular factors and processes guiding homeostatic control of the proteome (proteostasis) during AAI is essential to identify novel causes of improved stress resistance and nutritional health. Collectively protein synthesis, proteolysis, and targeting are central to proteostasis and AAI triggers stress signaling pathways that are central for cell adaptation. The overarching hypothesis of this proposal is that the outcome of these key signaling networks that respond to AAI are vital to controlling proteostasis and disease resistance. Cellular sensing of AAI involves overlapping signal transduction mechanisms that are largely conserved from yeast to humans. Our published and preliminary data demonstrate that key among these networks is the multi-part Integrated Stress Response (ISR), which features translational and transcriptional gene expression networks directed by phosphorylation of eukaryotic initiation factor 2. In the ISR network, the transcription factor ATF4 is central to directing the gene expression programs that help ameliorate AAI. The objective of this application is to define the contribution of the ISR to the early molecular and physiological responses that function to maintain proteostasis during dietary AAI. To achieve the objective of this application we propose three Specific Aims: 1) Define how variations in AAI activate the ISR; 2) Determine the role of ATF4 in the transcriptional networks and proteostatic responses to AAI; and 3) Assess novel control of proteolysis by the ISR during AAI. To accomplish these aims, time course studies will be conducted in cell lines and genetic strains of mice with targeted deficiencies in the ISR. Analyses will utilize a combination of sophisticated molecular biology and stable isotope techniques to assess and compare the mechanism of ISR activation and propagation in cell lysates and in the liver and skeletal muscle of mice during AAI. The proposal is innovative in that it will reveal for the first time how the transcriptome and translatome are guided by the ISR during AAI in vivo and provide insight into how the ISR coordinates with other nutrient sensing networks to regulate protein balance. The work proposed is significant because a greater understanding of the mechanisms activated by AAI will lead to new molecular targets and approaches to better prevent or treat human diseases.